The invention relates generally to the transformation of eukaryotic cells, particularly plant cells, with exogenous DNA and the generation of transgenic organisms, tissues or cultures from such cells.
Several methods have been developed for introducing exogenous DNA molecules into eukaryotic cells in order to take advantage of the widespread benefits arising from the application of recombinant DNA technology to the production of transgenic organisms. These methods include physical, non-biological systems such as electroporation, microinjection, calcium phosphate or polyethylene glycol (PEG) mediated DNA uptake or cell fusion, and microprojectile bombardment (aka xe2x80x9cbiolisticsxe2x80x9d) and modified biological systems such as Agrobacterium-mediated T-DNA transfer to plant cells (for a general and somewhat dated overview, see chapters 2 and 3 of xe2x80x9cPlant Genetic Transformation and Gene Expression, A Laboratory Manualxe2x80x9d, ed. by Draper, J. et al., pub. by Blackwell Scientific Publications (1988); see also Potrykus, et al., xe2x80x9cDirect Gene Transfer: State of the Art and Future Potentialxe2x80x9d, Plant Mol. Biol. Rep. 3: 117-128 (1985)).
The methods which have been developed have allowed the stable transformation of a wide variety of organisms with exogenous DNA. In particular, the development of physical techniques such as microprojectile bombardment has overcome apparent host-range limitations imposed by biological systems. However, a common deficiency of these physical methods is that they do not provide any means for ordered integration of the delivered DNA into the cell genome. Consequently these methods must depend upon uncontrolled integration of the delivered DNA by poorly understood mechanisms, causing exogenous DNA to be integrated as multiple copies of random fragments usually at a single site in the cell genome.
Improving the predictability of stable transformation events arising from the physical introduction of exogenous DNA into the cell would significantly improve the utility and overall efficiency of these processes for producing genetically stable transformed organisms exhibiting stable expression of transgenes. One approach which has been taken to accomplish this goal has been to combine proteins which promote transformation and/or integration in biological systems with non-biological delivery techniques. In order to achieve the desired effect, it has been considered necessary to associate the proteins themselves with the exogenous DNA molecules in advance of delivery to the transformation target cell, thus mimicking as closely as possible the biological system from which the proteins are derived (see, e.g. international application no. PCT/EP94/02566 to Hohn et al. published Feb. 23, 1995 as WO 95/05471; international application no. PCT/US95/07543 to Conary, J. et al. published Dec. 21, 1995 as WO 95/34647).
The present invention provides an improved method for stably transforming eukaryotic cells with exogenous DNA. This improved method generally comprises providing to the eukaryotic cell targeted for transformation at least one chimeric gene or mRNA capable of producing one or more proteins that promote integration in combination with the exogenous DNA desired to be integrated.
In particular, the present invention provides an improved method for stably transforming plant cells with exogenous DNA, which combines positive attributes of Agrobacterium tumefaciens mediated T-DNA transfer and integration with non-biological delivery methods. This improved method comprises providing a plant cell with the exogenous DNA fragment desired to be integrated into the plant cell genome, bounded by T-DNA borders, along with at least one chimeric gene or RNA capable of expressing, in the plant cell, an Agrobacterium-derived protein that promotes the integration of the exogenous DNA. The Agrobacterium-derived protein provided according to the invention particularly includes VirD1, VirD2, and VirE2. Preferably, VirD2 and VirD1, either alone or in combination with VirC and/or VirE2, or a subcombination thereof, is used. Expression of the Agrobacterium-derived protein(s) in the plant cell causes the integration of the exogenous DNA as an intact fragment with predictable endpoints.
According to the invention, the exogenous DNA fragment bounded by T-DNA border sequences may be delivered to the plant cell or other eukaryotic cell by non-biological means such as, but not restricted to, electroporation, microinjection, induced uptake, microprojectile bombardment, or other means as are known in the art.
According to the invention, the Agrobacterium-derived protein(s) may also be delivered to the plant cell or other eukaryotic cell by non-biological means in the form of DNA (chimeric gene expressible in the cell) or RNA (RNA translatable in the cell).
The exogenous DNA fragment and the Agrobacterium-derived protein(s) in the form of DNA or RNA are temporally delivered so that the Agrobacterium-derived protein(s) are present in the plant cell or other eukaryotic cell after the exogenous DNA has been delivered and before the exogenous DNA has been integrated. This may preferably be achieved by simultaneous delivery of these components in a single step. Alternatively, the cell targeted for transformation may be derived from an organism or cell culture that has previously been stably transformed with a chimeric gene(s) capable of expressing the Agrobacterium-derived protein(s).
In another aspect of the invention, eukaryotic cells stably transformed with a discrete DNA fragment are regenerated to produce fertile transgenic organisms that stably express a desired transgene and pass it on to progeny in which stable expression of the transgene is inherited as a Mendelian trait.